Project Summary/Abstract The goal of this renewal application is to elucidate the impact of the lipid membrane enclosing cellular cargos on the function of the major microtubule-based motor protein kinesin-1. Motor protein-based motility underlies many physiologically important processes, including the delivery of vesicular cargos from one subcellular location to another in neurons. Dysfunctions in this intracellular motility are implicated in many diseases, including neurodegeneration. While the properties of motor proteins have been extensively studied both in vivo and in vitro, many important questions remain, including how the properties of the cargo itself impact motor function. The central hypothesis of this proposal is that the fluid nature of the cargo membrane and the formation of microdomains in the membrane are key regulators of motor protein-based motility. In cells, motor proteins often work in small teams to move membrane-bound, cargo-containing vesicles. Using traditional in vitro cargos that lack an enclosing membrane, the motility of the cargo are shown to correlate positively with the number of motors actively moving that cargo. The importance of the cargo membrane in determining the number of motors in a team has long been proposed. First, membrane fluidity can enable the redistribution and clustering of motor proteins near the microtubule. Second, membrane microdomains can serve as preferential binding sites that cluster motors. Both mechanisms can increase the number of motors that are available to move the cargo as a team. Crucially, most cargos in current in vitro assays still lack the physiological membrane. Thus, quantitative investigations of these proposed mechanisms are limited by a lack of appropriate in vitro experimental systems. To close this major gap, during the current funding period, the research team combined advances in membrane biophysics with established single-molecule optical trapping to characterize the motility of membrane-enclosed cargos in vitro. Using this new in vitro experimental system, the research team uncovered the first direct link that the presence of a fluid membrane positively impacts the motility of the major microtubule-based motor protein kinesin. Preliminary analyses further indicate that the increase in cargo motility correlates with an increase in the number of kinesins moving the cargo. Together, this recent work lays the foundation for the next funding period, when the research team will directly test the central hypothesis that cargo-membrane fluidity and microdomain formation are key regulators of motor protein-based motility. Accomplishing the proposed Aims will establish the in vitro system used in this proposal as a controlled experimental platform for interrogating the physiological regulation of motor proteins. Findings of the proposed studies have the potential to shed light on the molecular mechanisms underlying diseases, including neurodegeneration. Both new investigations and novel therapeutic targets and strategies to mitigate neurological pathology and to promote cellular health will arise from the studies proposed here.